Vascular permeability factor/vascular endothelial growth factor-mediated signaling in mouse mesentery vascular endothelium

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a multifunctional cytokine and growth factor that has important roles in both pathological and physiological angiogenesis. VPF/VEGF induces vascular hyperpermeability, cell division, and other activities by interacting with two specific receptor tyrosine kinases, KDR/Flk-1 and Flt-1, that are selectively expressed on vascular endothelium. The signaling cascade that follows VPF/VEGF interaction with cultured endothelium is only partially understood but is known to result in increased intracellular calcium, activation of protein kinase C, and tyrosine phosphorylations of both receptors, phospholipase C-gamma (PLC-gamma) and phosphatidylinositol 3'-kinase. For many reasons, signaling events elicited in cultured endothelium may not mimic mediator effects on intact normal or tumor-induced microvessels in vivo. Therefore, we developed a system that would allow measurement of VPF/VEGF-induced signaling on intact microvessels. We used mouse mesentery, a tissue whose numerous microvessels are highly responsive to VPF/VEGF and that we found to express Flk-1 and Flt-1 selectively. At intervals after injecting VPF/VEGF i.p., mesenteries were harvested, extracted, and immunoprecipitated. Immunoblots confirmed that VPF/VEGF induced tyrosine phosphorylation of several proteins in mesenteric microvessels as in cultured endothelium: Flk-1; PLC-gamma; and mitogen-activated protein kinase. Similar phosphorylations were observed when mesentery was exposed to VPF/VEGF in vitro, or when mesenteries were harvested from mice bearing the mouse ovarian tumor ascites tumor, which itself secretes abundant VPF/VEGF. Other experiments further elucidated the VPF/VEGF signaling pathway, demonstrating phosphorylation of both PYK2 and focal adhesion kinase, activation of c-jun-NH2-kinase with phosphorylation of c-Jun, and an association between Flk-1 and PLC-gamma. In addition, we demonstrated translocation of mitogen-activated protein kinase to the cell nucleus in cultured endothelium. Taken together, these experiments describe a new model system with the potential for investigating signaling events in response to diverse mediators on intact microvessels in vivo and have further elucidated the VPF/VEGF signaling cascade.     

Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, induces endothelial proliferation in vitro and vascular permeability in vivo. The human transmembrane c-fms-like tyrosine kinase Flt-1 has recently been identified as a VEGF receptor. Flt-1 kinase has seven immunoglobulin-like extracellular domains and a kinase insert sequence, features shared by two other human gene-encoded proteins, kinase insert domain-containing receptor (KDR) and FLT-4. In this study we show that the mouse homologue of KDR, Flk-1, is a second functional VEGF receptor. Flk-1 binds VEGF with high affinity, undergoes autophosphorylation, and mediates VEGF-dependent Ca2+ efflux in Xenopus oocytes injected with Flk-1 mRNA. We also demonstrate by in situ hybridization that Flk-1 protein expression in the mouse embryo is restricted to the vascular endothelium and the umbilical cord stroma. VEGF and its receptors Flk-1/KDR and Flt-1 may play a role in vascular development and regulation of vascular permeability.     

Reduced intracellular oxidative metabolism promotes firm adhesion of human polymorphonuclear leukocytes to vascular endothelium under flow conditions

The interaction of polymorphonuclear leukocytes (PMN) with the vascular endothelium and their subsequent extravasation to the tissues is a key step during different physiological and pathological processes. In certain of these pathologies the oxygen tension becomes very low, leading to reduced cellular oxidative status. To evaluate the effect of lowering the intracellular redox status in the interaction of PMN with the endothelium, exposure to hypoxic conditions as well as treatment with different antioxidant agents was carried out. PMN exposure to hypoxia enhanced beta2 integrin-dependent adhesion to intercellular adhesion molecule-1-coated surfaces, concomitant with a decrease in the intracellular redox status of the cell. As occurs with hypoxia, treatment with antioxidants produced a decrease in the oxidation state of PMN. These agents enhanced adhesion of PMN to human umbilical vein endothelial cells stimulated with tumor necrosis factor-alpha (TNF-alpha), and this effect was also mediated by beta2 integrins LFA-1 and Mac-1. Adhesion studies under defined laminar flow conditions showed that the antioxidant treatment induced an enhanced adhesion mediated by beta2 integrins with a decrease in the fraction of PMN rolling on TNF-alpha-activated endothelial cells. The up-regulated PMN adhesion was correlated to an increase in the expression and activation of integrin Mac-1, without loss of L-selectin surface expression. Altogether, these results demonstrate that a reduction in the intracellular oxidative state produces an enhanced beta2 integrin-dependent adhesion of PMN to stimulated endothelial cells under conditions of flow.     

Vascular adhesion protein 1 (VAP-1) mediates lymphocyte subtype-specific, selectin-independent recognition of vascular endothelium in human lymph nodes

Interactions between lymphocyte surface receptors and their ligands on vascular endothelial cells regulate the exit of lymphocytes from the circulation. Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized. Here we show that vascular adhesion protein-1 (VAP-1) mediates subtype-specific binding of CD8-positive T cells and natural killer cells to human endothelium. VAP-1-dependent, oligosaccharide-dependent peripheral lymph node (PLN) HEV adhesion under shear was independent of L-selectin, P-selectin glycoprotein ligand 1, and alpha4 integrins, the known lymphocyte receptors involved in the initial recognition of endothelial cells. PLN HEV adhesion was also critically dependent on peripheral lymph node vascular addressins (PNAds), but lymphocyte L-selectin was absolutely required for PNAd binding. Most lymphocytes relied on both PNAd and VAP-1 in HEV binding. The overlapping function of L-selectin ligands and VAP-1 in PLN introduces a new control point into the lymphocyte extravasation process. Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo. In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.     

Endocardial endothelium selectively modifies relaxation in rat papillary muscle

The selective removal of endocardial endothelium of rat left ventricular papillary muscles by 1-second immersion in 0.5% Triton X-100 showed little influence on resting tension and only a small decrease in peak isometric tension (8.3 +/- 1.4 vs 9.6 +/- 2.4 mN/mm2 at Lmax, p > 0.05) with no reduction in maximal rate of tension development (+dT/dtmax; 136 +/- 21 vs 137 +/- 18 mN/mm2/s, p > 0.05). In contrast, there was a marked increase in maximal rate of tension decline (-dT/dtmax) from 71 +/- 14 to 92 +/- 15 mN/mm2/s (p < 0.05), so that the ratio between +dT/dtmax and -dT/dtmax fell from 1.98 +/- 0.27 to 1.51 +/- 0.13 (p < 0.01). Removal of endocardial endothelium led to a significant shortening of isometric twitch contractions. Time to peak tension was abbreviated from 111 +/- 20 to 84 +/- 8 ms (p < 0.05) and the half relaxation time from 92 +/- 9 to 68 +/- 8 ms (p < 0.01). Time to +dT/dtmax was also shortened from 31 +/- 6 to 44 +/- 9 ms (p < 0.05) and time to -dT/dtmax from 90 +/- 12 to 62 +/- 10 ms (p < 0.01). These effects were not influenced by alterations in stimulation frequency or muscle length. The early onset of relaxation and abbreviated duration of relaxation together with an increased rate of decline in tension led to a shorter total twitch which may explain the slightly lower peak tension once the endocardial endothelium was removed. Our findings confirm that endocardial endothelium modulates myocardial contraction, with a predominant influence on relaxation.     

Vascular endothelium: an integrator of pathophysiologic stimuli in atherosclerosis

Vascular endothelium, the single-cell-thick lining of the cardiovascular system, is an important functional component of the blood vessel wall, actively participating in normal vascular physiology as well as the pathogenesis of vascular diseases such as atherosclerosis. The localized modulation of vascular endothelium to a non-adaptive functional state can be termed "endothelial dysfunction." This article provides a brief overview of endothelial dysfunction, especially as it relates to mononuclear leukocyte recruitment during atherosclerotic lesion formation. Potential diagnostic and therapeutic implications are also considered.     

The vascular endothelium: physiopathology and participation in thrombogenesis

Even though homeothermic animals regulate the body temperature, fluctuations up to 2-3 degrees C may occur during physiological conditions. In many species, including the rat, a similar variation can be measured in the brain temperature. Such changes are expressed throughout the brain with a preserved gradient between the warmer basal and cooler dorsal parts. In spite of these recordable physiological changes, spatial learning is quite robust, in that it occurs at brain temperatures between 30 and 39 degrees C. Even drastic cooling (to below 15 degrees C) fails to affect consolidation or storage of information when the animal is tested after rewarming. The physiological temperature fluctuations have significant consequences for electrophysiological responses in the brain. Various bioelectrical signals are more sensitive during warming, axonal conduction is speeded up, and stimulus-elicited transmitter release becomes faster and more synchronized. Action potentials have shorter rise and decay times in warm conditions, and the amplitude becomes slightly smaller. Population responses are differently affected by these changes. Dentate field potentials in response to stimulation of perforant-path fibers appear with shorter latency in warm conditions, and the rate of rise in the field EPSP is increased. Paradoxically, the amplitude of the population spike is reduced. This is due to a combination of reduced amplitude of individual action potentials and reduced efficiency of the summation of groups of action potentials. Due to the large effects of temperature on hippocampal field potentials, it is mandatory that brain temperature changes are monitored and/or controlled whenever such responses are recorded in freely moving and anesthetized animals.     

Chromosomal mapping of two novel human FGF genes, FGF11 and FGF12

The fibroblast growth factor (FGF) family comprises to date 12 members, which are involved in various physiological processes throughout embryogenesis and adult life. Two novel members of the family have been identified recently (FGF11 and FGF12). Using in situ hybridization on metaphasic chromosomes, we have been able to assign FGF11 to band p12-p13 of human chromosome 17 and FGF12 to band q28 of human chromosome 3.     

The role of adhesion molecules in human leukocyte attachment to porcine vascular endothelium: implications for xenotransplantation

Many obstacles still prevent successful xenotransplantation of porcine donor organs. When hyperacute rejection is averted, transplanted pig organs are subject to acute vascular and cellular rejection. In autologous systems, leukocyte recruitment into inflamed tissues involves selectins, integrins, and Ig family members. To determine whether these mechanisms allow human leukocytes to effectively enter porcine grafts, the pathways by which human leukocytes adhere to TNF-alpha-stimulated porcine aortic endothelium were examined under static and physiologic flow conditions. L-selectin and E-selectin had overlapping functions in neutrophil capture and rolling, whereas Ab blockade of E-selectin and the beta2 integrins inhibited firm arrest of rolling neutrophils. Combined blockade of selectins and beta2 integrins resulted in negligible human neutrophil attachment to pig endothelium. Lymphocyte attachment to porcine endothelium was primarily L-selectin mediated, whereas beta2 integrin and VCAM-1/very late Ag-4 (VLA-4) interactions promoted static adhesion. Concurrent beta2 integrin, VLA-4, VCAM-1, and L-selectin blockade completely inhibited lymphocyte attachment. Thus, interactions between leukocyte-endothelial cell adhesion receptor pairs remained remarkably intact across the human-porcine species barrier. Moreover, disrupting the adhesion cascade may impair the ability of human leukocytes to infiltrate a transplanted porcine organ during rejection.     

Cell adhesion and short-term patency in human endothelium preseeded 1.5-mm polytetrafluoroethylene vascular grafts: an experimental study

It has been shown that endothelialization improves short-term patency of 1.5-mm expanded polytetrafluoroethylene vascular grafts. A model for endothelialization of 1.5-mm expanded polytetrafluoroethylene vascular grafts with human endothelial cells is described. In this model, the adherence of endothelial cells was increased significantly in grafts coated with serum proteins and collagen. By means of this protocol, graft patency was tested after implantation in two animal models: the rat aorta and the rabbit common carotid artery. Anastomosis was performed with a 3M Precise Microvascular Anastomotic System. In both animal models, no significant loss of endothelial cells in the precoated grafts (rat, n = 8) were noted 1 hour after blood flow restoration. All uncoated grafts showed significant endothelial cell loss. In the rabbit model, all nonendothelialized grafts (n = 8) clotted 5 to 25 minutes after flow restoration. Seven (n = 8) endothelialized grafts showed no clotting during 1 hour's observation: one clotted immediately for a patency rate of 87.5 percent. These results indicate that endothelialization of 1.5-mm grafts is practical. Furthermore, adhesion of endothelial cells to the graft walls is not affected by short-term, pulsatile, high-pressure blood flow.     

Sarcoplasmic reticulum genes are selectively down-regulated in cardiomyopathy produced by doxorubicin in rabbits

The clinical utility of doxorubicin, an antineoplastic agent, is limited by its cardiotoxicity. Our objective was to determine whether expression of genes encoding proteins that affect Ca2+ homeostasis were altered in the hearts of rabbits chronically treated with doxorubicin. Twelve male New Zealand white rabbits received an injection of doxorubicin (2.5 mg/kg i.v.) once a week for 8 weeks. Eight rabbits were similarly injected with saline as controls. The cardiac function of both groups was evaluated 8 weeks after the final injection, as were the levels of expression of mRNA for Ca2+ transport proteins in the sarcoplasmic reticulum and plasma membrane. The amount of the sarcoplasmic reticulum Ca2+-ATPase and the Ca2+ uptake capacity of the protein were also quantitated. Cardiac output was significantly decreased in the doxorubicin-treated group (71+/-21 ml/min, P<0.05) compared with the control group (118+/-15 ml/min). The mRNA levels for the sarcoplasmic reticulum proteins were significantly diminished in the doxorubicin-treated hearts: ryanodine receptor-2 (relative expression level compared with controls, 0.35+/-0.13, P<0.01), sarcoplasmic reticulum Ca2+-ATPase (0.56+/-0.13, P<0.01), phospholamban (0.62+/-0.20, P<0.01) and cardiac calsequestrin (0. 57+/-0.26, P<0.01). In addition, both relative amount of sarcoplasmic reticulum Ca2+-ATPase protein (doxorubicin-treated group, 69+/-17% of control, P<0.01) and the Ca2+ uptake capacity (46. 9+/-9.8 nmol Ca2+/mg protein-5 min in doxorubicin group v 63.2+/-10. 4 in the control group, P<0.01) were concomitantly decreased with its mRNA expression level. Conversely, the mRNA levels for the plasma membrane proteins did not differ from those of control rabbits: the dihydropyridine receptor (relative expression level, 1. 03+/-0.30, N.S.), plasma membrane Ca2+-ATPase (0.93+/-0.33, N.S.) and the Na+/Ca2+ exchanger (0.87+/-0.34, N.S.). These findings suggest that a selective decrease in mRNA expression for sarcoplasmic reticulum Ca2+ transport proteins is responsible for the impaired Ca2+ handling, and thus, for the reduced cardiac function seen in the cardiomyopathy induced in rabbits by the long-term treatment with doxorubicin.     

Identification of two closely related genes, inducible and noninducible carbonyl reductases in the rat ovary

OBJECTIVE: To investigate the potential role of cytokines in psoriatic arthritis (PsA) by assessing the profiles of the proinflammatory cytokines in synovial fluid (SF) of PsA in comparison with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 1 (IL-1), IL-6, and IL-8 were measured in SF using ELISA. RESULTS: Levels of TNF-alpha, IL-1beta, and IL-8 were significantly higher in PsA SF than in OA SF, although lower than in RA SF. No difference was detected in the IL-6 levels between PsA and RA SF, both of which were much higher than in OA SF. CONCLUSION: The pattern of expression of proinflammatory cytokines seen in PsA is similar to that in RA. Since PsA is also a destructive arthropathy, cytokines, in particular TNF-alpha and IL-1, may be principle factors in joint destruction.     

Identification of two novel diurnal genes by screening of a rat brain cDNA library

While the hypothalamus is fundamental for sleep and circadian regulation, the molecular mechanisms involved are poorly understood. We have used a differential gene expression technique to identify hypothalamic genes which have altered expression in rat sleep periods. Complex cDNA probes from rat hypothalami removed at Zeitgeber times 4 and 15 were hybridised to rat brain cDNA library girds. From 30 differentially expressed clones, six were further analysed and two were confirmed to exhibit increased expression at Zeitgeber time 4. A Northern blot hybridization of brain, heart, kidney, lung, testis and skin mRNA showed that both clones were brain specific. Therefore, we have identified two novel brain specific diurnally expressed hypothalamic genes. Both genes may have roles in sleep or circadian regulation.     

Complement-induced expression of chemokine genes in endothelium: regulation by IL-1-dependent and -independent mechanisms

Activation of complement in the vicinity of endothelium is thought to contribute to the tissue manifestations of inflammatory and immune responses. Endothelial cells contribute to these processes in part by the elaboration of chemokines that activate various leukocytes and direct their migration into tissues. We investigated the mechanisms by which activation of complement on endothelial cell surfaces might influence the expression of chemokine genes in endothelial cells. In a model for the immune reaction occurring in a xenograft, human serum, as a source of xenoreactive anti-endothelial Abs and complement, induced expression of the monocyte chemotactic protein-1 (MCP-1), IL-8, and RANTES genes. The MCP-1 and IL-8 genes were expressed within 3 h as a first phase and at > 12 h as a second phase. The RANTES gene was expressed in porcine endothelial cells only 12 h after exposure to human serum. The expression of these genes required activation of complement and assembly of membrane attack complex, as it was inhibited by soluble CR1 and did not occur in the absence of C8. The early phase of MCP-1 and IL-8 gene expression did not require de novo protein synthesis. The late phase of MCP-1, IL-8, and RANTES gene expression predominantly required the production of IL-1alpha as an intermediate step. The results indicate that the expression of chemokine genes in endothelial cells occurs as a function of differential responses to complement and may in part be conditioned by the availability of IL-1alpha.     

Cardiac allograft vasculopathy: IgM antibody responses to donor-specific vascular endothelium

Stunning and slaughter trials were conducted to evaluate the influence of carcass orientation (inverted or supine), angle of feather extraction (parallel or perpendicular to the carcass surface), and slaughter method (exsanguination without or with spinal cord transection) on feather retention force (FRF) in commercial broilers sampled ante-, peri-, and post-mortem. The pectoral, sternal, and femoral feather tracts were sampled before and after stunning contralaterally, with a maximum indicating force gauge, from broilers suspended on a shackle (inverted) or laying on a table (supine). For all trials and sample periods FRF was consistently greater in the femoral area (547 to 679 g) than in the pectoral area (273 to 391 g), with the sternal feather tract requiring the least force at 246 to 343 g. Feathers extracted parallel to the carcass resulted in consistently greater FRF (9 to 29%) than feathers extracted at a perpendicular angle, at all sample periods. Broilers suspended on shackles ante- and peri-mortem had higher FRF values (5 to 30%) than those restrained in shackles in a supine position on a table. Other parameters resulted in minor and inconsistent alterations in FRF. Electrical stunning, when not followed by bleeding, resulted in small reductions in FRF (up to 7%). Bleeding after stunning without or with spinal cord transection resulted in variable peri-mortem FRF changes (+7 to -11% and +11 to -11%, respectively). Only in the pectoral feather tract was there a significant increase (5 to 6%) in FRF as broilers went from the ante- to peri-mortem state. At 2 and 6 min after stunning and initiation of exsanguination, post-mortem FRF was unaffected by carcass orientation for the pectoral and femoral tracts.     

Signaltransduction in vascular endothelium: the role of intracellular calcium and ion channels

Endothelial cells (ECs) provide an ideal surface for blood flow. They inhibit the initiation of blood-clotting, but can also under certain conditions activate this process. ECs influence thrombolysis as well as thrombogenesis. They are "antigen-presenting cells" and play a key role in angiogenesis. In addition, ECs control the permeability of the barrier between bloodvessels and interstitium. One of their most important functions is the regulation of the diameter of the blood vessels and their adaptation to the demanded hemodynamic needs. The production and release of diverse compounds, which interfere with different neighboured target cells, initiate this plethora of functions. Ca2+ signals in endothelial cells play the key role in the release of NO, prostacyclin (PGI2), platelet activating factor (PAF), von Willebrand factor (vWF), tissue plasminogen activator (tPA) and tissue factor pathway inhibitor (TFPI). Changes in the intracellular Ca2+ concentration ([Ca2+]i) are determined by release from intracellular stores and entry through the plasma membrane. The diversity of Ca2+ entry pathways and mechanisms of their control are described. At least two different types of Ca2+ entry channels exist: 1. typical highly Ca2+ selective ion channels which might be activated by depletion of intracellular Ca2+ stores (Ca2+ release-activated Ca2+ channels, CRAC), and 2. Non-selective Ca2+ permeable cation channels (NSC). The latter shares many features with an NSC induced by expression of the protein TRPC3. These channels are only weakly operated by store depletion and require a permissive Ca2+ and Ins(1,4,5)P3 concentration in the cytosol. CRAC channels are possible indirectly involved in Ca2+ entry during mechano-stimulation of ECs. After activation of these entry channels, influx of Ca2+ depends on the driving force. The following ion channels play a pivotal role in regulation of the driving force for Ca2+ entry: an inwardly rectifying K+ channel, identified as Kir2.1, a large-conductance, Ca2+ activated K+ channel (hslo) and at least two Cl- channels (a volume regulated Cl- channel, VRAC, and a Ca2+ activated Cl- channel, CaCC). It will be explained how these ion channels interact in the regulation of the long-lasting (plateau-type) increase in [Ca2+]i which mainly controls NO-synthesis and release. Furthermore, it will be demonstrated that Ca2+ oscillations depend on intracellular events rather than Ca2+ entry from the extracellular space.